Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/07—Animals genetically altered by homologous recombination
  • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
  • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
  • G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH

Definitions

• This invention relates to a novel G-protein coupled glycoprotein hormone receptor in substantially purified form, and also to mutant or polymorphic forms of the receptor, recombinant nucleic acids encoding the same, recombinant host cells transformed with the nucleic acids, transgenic knockout animals lacking the receptor, transgenic animals expressing a non-native receptor gene, antibodies against the receptor and polypeptides thereof, and the uses of the receptor, recombinant nucleic acids, recombinant host cells and transgenic animals in drug screening and development, diagnosis and therapeutic applications.
• the G-protein coupled receptor of the present invention is a member of the glycoprotein hormone receptor family. Only three G- protein coupled glycoprotein hormone receptors have been previously reported: the Follicle Stimulating Hormone (FSH) Receptor (Minegish, et. al, 1991. Biomed. Biochem. Res. Comm. 175:1125-1130; Sprengel, et. al, 1990. Mol. Endocrinol. 4:525-530); the Thyroid Stimulating Hormone (TSH) Receptor (Frazier, et. al, 1990. Mol. Endocrinol. 4:1264-1276; Parmentier, et. al, 1990.
• FSH Follicle Stimulating Hormone
• TSH Thyroid Stimulating Hormone
• glycoprotein hormone receptors exhibit a structure of the rhodopsin family G-protein coupled receptors.
• This class of receptors contains seven transmembrane domains with three extracellular loops and three intracellular loops.
• the large ligands, including the glycoprotein hormones, bind the N-terminal domain while smaller peptides, amines and other ligands can bind in a pocket formed by the extracellular loops.
• cytoplasmic loops particularly the third loop, and the C-terminal domain of the receptor are believed to interact with the G-protein.
• the receptor associated G-protein can be associated with several cellular signaling pathways. Most common are the adenylate- cyclase/cAMP pathway, the phospholipase C-b/phosphoinositol pathways and the elevation of intracellular Ca 2+ . These second messenger pathways mediate the action of the receptor ligand within the cell. They also advantageously can be used to assess the activity of a receptor in assays.
• Receptor activity can be regulated at the cellular level. Extensive activation of a receptor by agonists can result in phosphorylation of the C-terminus and cytoplasmic loops resulting in a rapid desensitization of the receptor. Further, receptors can be regulated by modulators of transcriptional activity on the receptor gene. cAMP responsive elements have been demonstrated within the promoter regions of some G-protein coupled receptor genes. Again, these aspects of cellular biochemistry can advantageously be used to monitor and assess receptor activity in assays, e.g., by monitoring receptor phosphorylation as an indication of the presence of an agonist of the receptor or monitoring transcriptional activity as an indication of the presence of a modulator of receptor gene expression.
• AOMF05 is expected to play an important role in the development and function of skeletal muscle, spinal cord, placenta, and, to a lesser extent, the brain.
• nucleic acid fragments of the AOMF05 G-protein coupled glycoprotein hormone receptor (SEQ ID NO:l) which encode a biologically active novel human receptor. Any such nucleic acid fragment will encode either a protein or protein fragment comprising at least an intracellular G-protein associating domain and/or extracellular ligand binding domain, domains conserved throughout the G-coupled glycoprotein hormone receptor family which exist in the amino acid sequence of AOMF05 variants a & b (SEQ ID NOS:2 & 4).
• any such polynucleotide includes but is not necessarily limited to nucleotide substitutions, deletions, additions, amino-terminal truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use, or would be useful for screening for modulators of expression, agonists and/or antagonists of AOMF05 function.
• the isolated nucleic acid molecule of the present invention can be a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which can be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide.
• DNA deoxyribonucleic acid molecule
• cDNA complementary DNA
• synthetic DNA such as a synthesized, single stranded polynucleotide.
• the isolated nucleic acid molecule of the present invention can also be a ribonucleic acid molecule (RNA).
• the nucleic acid can include the entire sequence of SEQ ID NOS:l or 3, a sequence encoding the open reading frame of SEQ ID NOS:l or 3, or smaller sequences useful for expressing peptides, or polypeptides of AOMF05 protein.
• the nucleic acid can have natural, non-natural or modified nucleotides or internucleotide linkages or mixtures of these.
• aspects of the present invention include nucleotide probes and primers derived from the nucleotide sequences disclosed herein as FIGS. 1A-1C, 3A-3F, 4A-4C, 6A-6F and SEQ ID NOS: 1, & 3.
• probes and primers are used to identify or isolate polynucleotides encoding AOMF05 or mutant or polymorphic forms of the AOMF05 receptor protein or gene.
• Probe and primers can be highly specific for AOMF05 nucleotide sequences.
• An aspect of this invention is a substantially purified form of the novel G-protein coupled glycoprotein hormone receptor protein, AOMF05, variant a, which is disclosed in FIG. 2 and as set forth in SEQ ID NO:2.
• An aspect of this invention is a substantially purified form of the novel G-protein coupled glycoprotein hormone receptor protein, AOMF05, variant b, which is disclosed in FIG. 8 and as set forth in SEQ ID NO:4.
• Aspects of the present invention include biologically active fragments and or mutants of an AOMF05 protein, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for modulators, agonists and/or antagonists of AOMF05 function.
• the fragment is a soluble N- terminal fragment that can compete with the receptor for receptor ligands.
• aspects of the present invention include recombinant vectors and recombinant hosts which contain the nucleic acid molecules disclosed throughout this specification.
• the vectors and hosts can be prokaryotic or eukaryotic.
• the hosts express AOMF05 peptides, polypepetides, proteins or fusion proteins.
• the host cells are used as a source of expression products.
• aspects of the invention are polyclonal and monoclonal antibodies raised in response to either the entire human form of AOMF05 disclosed herein, or only a fragment, or a single epitope thereof.
• antibodies are raised against epi topes within the NH 2 -terminal domain of AOMF05.
• antibodies are rasied to epitopes that are unique to the AOMF05 receptor.
• An Aspect of this invention is the use of the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention to screen and measure levels of human AOMF05.
• the recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of human AOMF05.
• aspects of this invention are assays to detect agonists and antagonists of the AOMF05 receptor and modulators of the expression of AOMF05.
• cells comprising AOMF05 are used in screening assays including the melanophore system, yeast expressing mammalian adenylate cyclase, yeast pheromone protein surrogate screening, phospholipase second signal screening and the yeast two-hybrid system, all of which are well known and simply adapted by one of skill in the art.
• An aspect of this invention is tissue typing using probes or antibodies of this invention.
• polynucleotide probes are used to identify tissues expressing AOMF05 RNA.
• probes or antibodies can be used to identify a type of tissue based on AOMF05 expression or display of AOMF05 receptors on the surface of one or more cells.
• An aspect of this invention is isolated nucleic acid molecules which are fusion constructions expressing fusion proteins useful in assays to identify compounds which are modulators, agonist or antagonists of wild-type human AOMF05 activity.
• a preferred embodiment of this aspect of the invention includes, but is not limited to, glutathione S-transferase GST-AOMF05 fusion constructs. These fusion constructs include, but are not limited to, all or a portion of the ligand- binding domain of AOMF05, as an in-frame fusion at the carboxy terminus of the GST gene.
• the fusion protein is useful to isolate or identify ligands of the AOMF05 receptor.
• SEQ ID NOS:l-4 allow the artisan of ordinary skill to construct any such nucleic acid molecule encoding a GST-G-protein coupled glycoprotein hormone receptor fusion protein.
• Soluble recombinant GST-G-protein coupled glycoprotein hormone receptor fusion proteins can be expressed in various expression systems, including Spodoptera frugiperda (Sf21) insect cells (Invitrogen) using a baculovirus expression vector (e.g., Bac- N-Blue DNA from Invitrogen or pAcG2T from Pharmingen).
• compositions including an AOMF05 protein, fragments thereof, agonists, antagonists or modulators of AOMF05 or AOMF05 polynucleotides.
• An aspect of this invention is using polynucleotides according to the invention in methods of gene therapy, for instance in treatment of individuals with the aim of preventing or curing (wholly or partially) disease states associated with mutations in the AOMF05 gene. This may ease one or more symptoms of the disease.
• Introduction of nucleic acid may take place in vivo by way of gene therapy vectors and methods.
• An aspect of this invention is a transgenic animal useful for the study of the tissue and temporal specific expression or activity of the AOMF05 receptor in a non-human animal.
• the animal is also useful for studying the ability of a variety of compounds to act as modulators of AOMF05 receptor activity or expression in vivo or, by providing cells for culture or assays, in vitro.
• the animal is used in a method for the preparation of a further animal which lacks a functional endogenous AOMF05 gene.
• the animal of this aspect is used in a method to prepare an animal which expresses a non-native AOMF05 gene in the absence of the expression of a endogenous gene.
• the non-human animal is a mouse.
• the non-native AOMF05 gene is a wild-type human gene or a mutant human AOMF05 gene.
• FIGS. 1A-1B Schematically depicts the nucleotide sequence of a cDNA polynucleotide encoding the AOMF05 receptor, variant a (SEQ ID NO:l).
• FIG. 2 Schematically depicts the full length amino acid sequence of the AOMF05 receptor protein, variant a, (SEQ ID NO:2) in single letter code.
• FIGS. 3A-3F Schematically depicts the nucleotide sequence of a polynucleotide encoding AOMF05 (nucleotides 2-3950 of SEQ ID NO:l) and the translation of the AOMF05 open reading frame (SEQ ID NO:2).
• FIGS. 4A-4B Schematically depicts the nucleotide sequence of a cDNA polynucleotide encoding the AOMF05 receptor, variant b (SEQ ID NO:3).
• FIG. 5. Schematically depicts the full length amino acid sequence of the AOMF05 receptor protein, variant b, (SEQ ID NO:4) in single letter code.
• FIGS. 6A-6F Schematically depicts the nucleotide sequence of a polynucleotide encoding AOMF05 (nucleotides 2-3950 of SEQ ID NO:3) and the translation of the AOMF05 open reading frame (SEQ ID NO:4).
• FIG. 7 Depicts nine predicted signal peptide cleavage sites of the AOMF05 protein. The nine sequences depicted are amino acids 7- 49, 557-599, 12-54, 5-47, 664-706, 634-675, 9-51, 666-708 and 553-595 of SEQ ID NO:2 respectively, in single letter code. The predicted cleavage sites apply to both variants a & b.
• FIG. 8 Depicts a Multi-tissue Northern blot analysis of the expression of the AOMF05 receptor gene.
• This invention provides polynucleotides and polypeptides of a human G-coupled glycoprotein hormone receptor, referred to herein as AOMF05.
• the polynucleotides and polypeptides are used to further provide expression vectors, host cells comprising the vectors, non- human animals transgenic for the polynucleotides, knockout animals, probes and primers, antibodies against the receptor and polypeptides thereof, assays for the presence or expression of AOMF05 and assays for the identification of modulators, agonists and antagonists of the AOMF05 receptor.
• the AOMF05 gene, receptor and agonists, antagonists and modulators thereof can be useful in the treatment of diseases of the pancreas. Further uses include the treatment of obesity and diabetes. Further uses can include to stimulate the growth or regeneration of cells of the skeletal muscles.
• a "compound” or a "molecule” is an organic or inorganic assembly of atoms of any size, and can include macromolecules, e.g., peptides, polypeptides, whole proteins, and polynucleotides. The terms are used interchangeable herein.
• a “candidate” is a molecule or compound that may be an modulator, agonist or antagonist of an AOMF05 receptor.
• an "agonist” is a compound or molecule that interacts with and activates a polypeptide of an AOMF05 receptor.
• An activated AOMF05 receptor polypeptide can stimulate the cleavage of GTP by a G protein, activate the adenylate cyclase pathway or activate the phospholipase b pathway.
• an "antagonist” is a compound or molecule that interacts with and inhibits or prevents a polypeptide of an AOMF05 receptor from becoming activated.
• a modulator is a compound or molecule that interacts with an aspect of cellular biochemistry to effect an increase or decrease in the amount of a polypeptide of an AOMF05 receptor present at the surface of a cell, or in the surrounding serum or media.
• the change in amount of the receptor polypeptide can be mediated by the effect of a modulator on the expression of the receptor, e.g. , the transcription, translation, post-translational processing, translocation or folding of the receptor, or by affecting a component(s) of cellular biochemistry that directly or indirectly participates in the expression of the receptor.
• a modulator can act by accelerating or decelerating the turnover of the receptor either by direct interaction with the receptor or by interacting with another component(s) of cellular biochemistry which directly or indirectly effects the change.
• FIGS.1A-1C and SEQ ID NO:l a human cDNA encoding a G-protein coupled glycoprotein hormone receptor, AOMF05, disclosed as follows:
• a variant ofAOMF05 can be naturally occurring or mana-made.
• FIGS.4A-4C and SEQ ID NO:3 A most preferred aspect of the present invention is disclosed in FIGS.4A-4C and SEQ ID NO:3, a human cDNA encoding a G-protein coupled glycoprotein hormone receptor, AOMF05, disclosed as follows:
• the isolated nucleic acid molecule of the present invention can include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which can be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide.
• DNA deoxyribonucleic acid molecule
• cDNA complementary DNA
• synthetic DNA such as a synthesized, single stranded polynucleotide.
• the isolated nucleic acid molecule of the present invention can also include a ribonucleic acid molecule (RNA).
• the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
• a "polynucleotide” is a nucleic acid of more than one nucleotide.
• a polynucleotide can be made up of multiple polynucleotide units that are referred to by description of the unit.
• a polynucleotide can comprise within its bounds a polynucleotide(s) having a coding sequence(s), a polynucleotide(s) that is a regulatory region(s) and/or other polynucleotide units commonly used in the art.
• an "expression vector” is a polynucleotide having regulatory regions operably linked to a coding region such that, when in a host cell, the vector can direct the expression of the coding sequence.
• the use of expression vectors is well known in the art. Expression vectors can be used in a variety of host cells and, therefore, the regulatory regions are preferably chosen as appropriate for the particular host cell.
• a “regulatory region” is a polynucleotide that can promote or enhance the initiation or termination of transcription or translation of a coding sequence.
• a regulatory region includes a sequence that is recognized by the RNA polymerase, ribosome, or associated transcription or translation initiation or termination factors of a host cell. Regulatory regions that direct the initiation of transcription or translation can direct constitutive or inducible expression of a coding sequence.
• Polynucleotides of this invention contain full length or partial length sequences of the mammalian AOMF05 receptor gene. Polynucleotides of this invention can be single or double stranded.
• the polynucleotides can be a coding, "sense,” strand or a complementary, "antisense,” strand.
• Antisense strands can be useful as modulators of the receptor by interacting with RNA encoding the receptor. Antisense strands are preferably less than full length strands having sequences unique or highly specific for RNA encoding the receptor.
• the polynucleotides can include deoxyribonucleotides, ribonucleotides or mixtures of both.
• the polynucleotides can be produced by cells, in cell-free biochemical reactions or through chemical synthesis.
• Non-natural or modified nucleotides including inosine, methyl-cytosine, deaza-guanosine, etc.
• Natural phosphodiester internucleotide linkages can be appropriate.
• polynucleotides can have non-natural linkages between the nucleotides.
• Non-natural linkages are well known in the art and include, without limitation, methylphosphonates, phosphorothioates, phosphorodithionates, phosphoroamidites and phosphate ester linkages.
• Dephospho-linkages are also known, as bridges between nucleotides. Examples of these include siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, and thioether bridges.
• PNA Poly Nucleic Acid
• nucleic acids claimed herein can be present in whole cells or in cell lysates or in a partially purified or substantially purified form.
• a polynucleotide is considered purified when it is purified away from environmental contaminants.
• a polynucleotide purifed and isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
• the present invention also relates to a substantially purified and isolated form of the novel G-protein coupled glycoprotein hormone receptor protein, AOMF05.
• a preferred embodiment is a protein of the sequence which is shown in FIG. 2, set forth in SEQ ID NO:2, and disclosed as follows in single letter code:
• SEQIDNO:2 We refer to proteins and polypeptides having a sequence corresponding to the sequence shown above as Variant a' proteins and polypeptides.
• a more preferred embodiment is a protein of the sequence which is shown in FIG.5, set forth in SEQ ID NO:4, and disclosed as follows in single letter code:
• the present invention also relates to biologically active fragments and mutant or polymorphic forms of AOMF05 as set forth as SEQ ID NOS:2 & 4, including but not necessarily limited to amino acid substitutions, deletions, additions, amino terminal truncations and carboxy-terminal truncations such that these mutations provide for proteins or protein fragments of diagnostic, therapeutic or prophylactic use and would be useful for screening for modulators, agonists and/or antagonists of AOMF05 function.
• the biologically active fragment of AOMF05 is a soluble N-terminal fragment that can compete with the complete AOMF05 receptor for ligands of the receptor.
• soluble forms of receptors are well known in the art and can be derived from the polypeptides disclosed herein. It is preferred that soluble N-terminal fragments lack the signal sequence, that is that lack about the first 20 amino adds of SEQ ID NO:2 or 4. By “about” it is meant that the fragment need not lack exactly 20 amino acids as it is expected that deletion or removal of more or less can be useful. The important point is not so much the amount deleted but that the N-terminal fragment retains ligand binding activity.
• Any AOMF05 fragment can be simply tested for competition with the AOMF05 receptor using an antagonist assay described herein.
• the length can also vary. Soluble N-terminal fragments having the sequence of SEQ ID NO:2 or 4 up to but not including the seven hydrophobic domains are preferred. For example, it is preferred that soluble N-terminal fragments extend up to about amino add 539 of SEQ ID NOS:2 or 4. Again, this need not be an exact endpoint, as other appropriate endpoints can be determined by simple testing, e.g., for binding activity compared to the wild-type.
• polynucleotide and polypeptide sequences provided herein to isolate polynucleotides encoding naturally occurring forms of AOMF05, one of skill in the art can determine whether such naturally occurring forms are mutant or polymorphic forms of AOMF05 by sequence comparison. One can further determine whether the encoded protein, or fragments of any AOMF05 protein, is biologically active by routine testing of the protein of fragment in a in vitro or in vivo assay for the biological activity of the AOMF05 receptor.
• N-terminal or C-terminal truncations, or internal additions or deletions in host cells and test for their ability to stimulate the cleavage of GTP by a G protein, activate the adenylate cyclase pathway or activate the phospholipase b pathway.
• this invention is also directed to those DNA sequences encode RNA comprising alternative codons which code for the eventual translation of the identical amino acid, as shown below:
• the present invention discloses codon redundancy which can result in differing DNA molecules expressing an identical protein.
• a sequence bearing one or more replaced codons will be defined as a degenerate variation.
• mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
• DNA sequences coding for a peptide can be altered so as to code for a peptide having properties that are different than those of the naturally occurring peptide.
• Methods of altering the DNA sequences include but are not limited to site directed mutagenesis.
• altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
• a "biologically active equivalent” or “functional derivative” of a wild-type human AOMF05 possesses a biological activity that is substantially similar to the biological activity of the wild type human AOMF05.
• the term “functional derivative” is intended to include the “fragments,” “mutants,” “variants,” “degenerate variants,” “analogs” and “homologues” or to “chemical derivatives” of the wild type human AOMF05 protein.
• fragment is meant to refer to any polypeptide subset of wild-type human AOMF05.
• mutant is meant to refer to a molecule that may be substantially similar to the wild-type form but possesses distinguishing biological characteristics.
• Such altered characteristics include but are in no way limited to altered substrate binding, altered substrate affinity and altered sensitivity to chemical compounds affecting biological activity of the human AOMF05 or human AOMF05 functional derivative.
• variant is meant to refer to a molecule substantially similar in structure and function to either the entire wild-type protein or to a fragment thereof.
• a molecule is "substantially similar" to a wild-type human AOMF05-like protein if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino add sequences are not identical.
• analog refers to a molecule substantially similar in function to either the full-length human AOMF05 protein or to a biologically active fragment thereof.
• a "polymorphic" AOMF05 is an AOMF05 that is naturally found as an allele in the population at large.
• a polymorphic form of AOMF05 can have a different nucleotide sequence from the particular human AOMF05 allele disclosed herein.
• a polymorphic AOMF05 gene can encode the same or different amino acid sequence as that disclosed herein.
• some polymorphic forms AOMF05 will exhibit biological characteristics that distinguish the form from wild-type receptor activity, in which case the polymorphic form is also a mutant.
• a protein or fragment thereof is considered purified or isolated when it is obtained at a concentration at least about five-fold to ten-fold higher than that found in nature.
• a protein or fragment thereof is considered substantially pure if it is obtained at a concentration of at least about 100-fold higher than that found in nature.
• a protein or fragment thereof is considered essentially pure if it is obtained at a concentration of at least about 1000-fold higher than that found in nature.
• the AOMF05 receptor disclosed herein shows a tissue spedfic pattern of expression. Therefore, polynucleotides of this invention can be used as probes for tissue typing. Polynucleotide probes comprising full length or partial sequences of SEQ ID NOS:l or 3 can be used to determine whether a tissue expresses AOMF05 RNA. The temporal and tissue spedfic expression of AOMF05 RNA throughout an animal can also be studied using polynucleotide probes. The effect of modulators that effect the transcription of the AOMF05 receptor gene can be studied via the use of these probes.
• a preferred probe is a single stranded antisense probe having at least the full length of the coding sequence of AOMF05.
• probes that have less than the full length sequence, but at least 14 contiguous nucleotides, preferably at least 15 or 16 nucleotides and more preferably at least 20 contiguous nucleotides, wherein the nucleotide sequences are highly spedfic for AOMF05 DNA or RNA.
• a nucleotide probe is "highly spedfic" for AOMF05 DNA or RNA if one of skill in the art can use standard techniques to determine hybridization and washing conditions through which one can detect an AOMF05 encoding DNA in a Southern Blot of total human genomic DNA (digested with a restriction enzyme to an average size of about 4000 nucleotides) without visually detectable nonspedfic background hybridization.
• a probe is specific if one can detect the AOMF05 DNA despite any visually detectable nonspecific backgound hybridization that may be present.
• the identification of a sequence(s) for use as a specific probe is well known in the art and involves choosing a sequence(s) that is unique to the target sequence, or is spedfic or highly specific thereto.
• polynucleotides that are probes have at least about 14 nucleotides, more preferably at least about 20-25 nucleotides, and also preferably about 30 to 35 nucleotides or longer.
• the longer probes are believed to be more spedfic for AOMF05 genes and RNAs and can be used under more stringent hybridization conditions. Longer probes can be used but can be more difficult to prepare synthetically, or can result in lower yields from a synthesis.
• Examples of sequences within SEQ ID NOS:l & 3 that are useful as probes or primers are the AOMF05 series of primers given in Example 1. However, one skilled in the art will recognize that these are only a few of the useful probe or primer sequences that can be derived from SEQ ID NOS:l & 3.
• Polynucleotides having sequences that are unique or highly spedfic for AOMF05 can be used as primers in amplification reaction assays. These assays can be used in tissue typing as described herein. Additionally, amplification reactions employing primers derived from AOMF05 sequences can be used to obtain amplified AOMF05 DNA using the AOMF05 DNA of the cells as an initial template.
• the AOMF05 DNA so obtained can be a mutant or polymorphic form of AOMF05 that differ from SEQ ID NOS:l or 3 by one or more nucleotides of the AOMF05 open reading frame or sequences flanking the ORF. The differences can be associated with a non-defective naturally occurring allele or with a defective form of AOMF05.
• polynucleotides of this invention can be used in allelic identification of various AOMF05 genes or the detection of a defective AOMF05 gene.
• Probes can be labeled by any number of ways known in the art including isotopes, enzymes, substrates, chemiluminescent, electrochemiluminescent, biotin and fret pairs among many others.
• a probe so labeled can generate a detectable signal directly (e.g., isotopes), or upon hybridization (fret pairs), or indirectly after a chemical (e.g., luminescence) or biochemical reaction (e.g., enzyme-substrate) or after binding a strepavidin linked moiety that can generate a detectable signal direclty or indirectly.
• the labeling of probes and the generation of detectable signals are well known techniques in the art.
• a primer is specific for the amplification of AOMF05 sequences if one of skill in the art can use standard techniques to determine conditions under which an amplification reaction yields a predominant amplified product corresponding to the AOMF05 sequences.
• a primer is highly spedfic if no background amplification products are visually detectable.
• amplification reactions include Polymerase Chain Reaction and Reverse Transcriptase Polymerase Chain Reaction (See e.g. , PCR Primer, edited by C.W.Dieffenbach and G.S.Dveksler, (1995). Cold Spring Harbor
• the AOMF05 nucleotide and amino add sequences provided herein can be used to isolate and/or clone AOMF05 polynucleotides. Any of a variety of procedures can be used to clone AOMF05. These methods include, but are not limited to, (1) a RACE PCR cloning technique (Frohman, et al., 1988, Proc. Natl. Acad. Sci.85: 8998-9002). 5' and/or 3' RACE can be performed to generate a full length cDNA sequence. This strategy involves using gene-sperific oligonucleotide primers for PCR amplification of AOMF05 cDNA.
• These gene-specific primers are designed through identification of an expressed sequence tag (EST) nucleotide sequence which has been identified by searching any number of publicly available nucleic acid and protein databases; (2) direct functional expression of the AOMF05 cDNA following the construction of an AOMFO ⁇ -containing cDNA library in an appropriate expression vector system; (3) screening a AOMFO ⁇ -containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled degenerate oligonucleotide probe designed from the amino add sequence of the AOMF05 protein; (4) screening a AOMFO ⁇ -containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the AOMF05 protein.
• EST expressed sequence tag
• This partial cDNA is obtained by the specific PCR amplification of AOMFO ⁇ DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence known for other receptors which are related to the AOMFO ⁇ protein (e.g. , leutenizing, follicle-stimulating and thyroid stimulating hormone receptors); ( ⁇ ) screening an AOMFO ⁇ -containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the AOMF05 protein.
• AOMFO ⁇ -containing cDNA library constructed in a bacteriophage or plasmid shuttle vector with a partial cDNA encoding the AOMF05 protein.
• This strategy can also involve using gene- spedfic oligonucleotide primers for PCR amplification of AOMF05 cDNA identified as an EST as described herein; or (6) designing ⁇ ' and 3' gene specific oligonucleotides using SEQ ID NO:l as a template so that either the full length cDNA can be generated by known PCR techniques, or a portion of the coding region can be generated by these same known PCR techniques to generate and isolate a portion of the coding region to use as a probe to screen one of numerous types of cDNA and/or genomic libraries in order to isolate a full length version of the nucleotide sequence encoding AOMFO ⁇ .
• libraries can be useful for isolating a human AOMFO ⁇ -encoding DNA, a mammalian AOMFO ⁇ homologue, or mutant or polymorphic forms of AOMFO ⁇ receptor DNA or RNA.
• Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines other than human cells or tissue such as primate, murine, rodent, porcine and bovine cells or any other such vertebrate host which contains AOMFO ⁇ -encoding DNA.
• an AOMFO ⁇ gene can be isolated by oligonucleotide- or polynucleotide- based hybridization screening of a vertebrate genomic library, includ ng but not limited to primate, murine, rodent, pordne or bovine genomic libraries, as well as concomitant human genomic DNA libraries. It is readily apparent to those skilled in the art that suitable cDNA libraries can be prepared from cells or cell lines which express an AOMFO ⁇ receptor.
• the selection of cells or cell lines for use in preparing a cDNA library to isolate a AOMFO ⁇ cDNA can be done by first detecting cell associated AOMFO ⁇ receptors using an assay for AOMFO ⁇ , e.g., an assay using antibodies disclosed herein or a PCR assay using AOMFO ⁇ -specific primers.
• Preparation of cDNA libraries can be performed by standard techniques well known in the art. Well known cDNA library construction techniques can be found for example, in Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Complementary DNA libraries can also be obtained from numerous commercial sources, including but not limited to Clontech Laboratories, Inc., Palo Alto, CA, USA and Stratagene, Inc., La Jolla, CA, USA.
• DNA encoding AOMFO ⁇ can also be isolated from a suitable genomic DNA library. Construction of genomic DNA libraries can be performed by standard techniques well known in the art. Well known genomic DNA library construction techniques can be found in Sambrook, et al., supra.
• the amino add sequence or DNA sequence of AOMFO ⁇ or a homologous protein may be necessary. To accomplish this, the AOMFO ⁇ or a homologous protein can be purified, e.g. , through cross reaction with the anti- AOMFO ⁇ antibodies taught herein, and partial amino acid sequence(s) determined by automated sequenators.
• the linear sequence of two regions of 6 to 8 amino acids can be determined for the PCR amplification of a partial AOMFO ⁇ DNA fragment.
• the DNA sequences capable of encoding them are synthesized. Because the genetic code is degenerate, more than one codon can be used to encode a particular amino add, and therefore, the amino add sequence can be encoded by any of a set of similar, degenerate, DNA oligonucleotides. Only one member of the degenerate set will be identical to the AOMFO ⁇ sequence but others in the set will be capable of hybridizing to AOMFO ⁇ DNA even in the presence of DNA oligonucleotides with mismatches.
• mismatched DNA oligonucleotides can still suffidently hybridize to the AOMFO ⁇ DNA to permit identification and isolation of AOMFO ⁇ encoding DNA.
• nucleotide sequence of a region of an expressed sequence can be identified by searching one or more available genomic databases.
• Gene-spedfic primers can be used to perform PCR
• the appropriate nucleotide sequence for use in a PCR-based method can be obtained from SEQ ID NO:l, either for the purpose of isolating overlapping ⁇ ' and 3' PCR products for generation of a full-length sequence coding for AOMFO ⁇ , or to isolate a portion of the nucleotide sequence coding for AOMFO ⁇ for use as a probe to screen one or more cDNA- or genomic-based libraries to isolate a full-length sequence encoding AOMFO ⁇ or AOMFO ⁇ -like proteins.
• the AOMFO ⁇ full length cDNA of the present invention was generated by a method of cDNA screening called Reduced Complexity cDNA Analysis (RCCA).
• RCCA Reduced Complexity cDNA Analysis
• the first method is effective but laborious and slow while the latter method is fast but limited in effi ⁇ ency.
• This RACE protocol is hindered by limited length of extension due to the use of the entire cellular mRNA population in a single reaction. Since smaller fragments are amplified much more efficiently than larger fragments by PCR in the same reaction, PCR products obtained using the second method are often quite small.
• the RCCA method improves upon known methods of cDNA library screening by initially constructing and subdividing cDNA libraries followed by isolating ⁇ '- and 3'- flanking fragments by PCR. Since each pool is unlikely to contain more than one clone for a given gene which is low to moderately expressed, competition between large and small PCR products in one pool does not exist, making it possible to isolate fragments of various sizes.
• One definite advantage of the method as described herein is the efficiency, throughput, and its potential to isolate alternatively spliced cDNA forms.
• the RCCA process provides for rapid extension of a partial cDNA sequence based on subdividing a primary cDNA library and DNA amplification by polymerase chain reaction (PCR).
• a cDNA library is constructed with cDNA primed by random, oligo-dT or a combination of both random and oligo-dT primers and then subdivided into pools at approximately 10,000 -20,000 clones per pool ("superpools"). Each superpool is amplified separately and therefore represents an independent portion of the cDNA molecules from the original mRNA source. Samples from all the superpools are collected and transferred into 96-well plates.
• a partial cDNA sequence such as SEQ ID NO:l
• positive pools containing the partial cDNA sequence are first identified by PCR with a pair of primers complementary to the cDNA sequence.
• Each positive pool in the library contains an independent clone of the cDNA sequence; within each clone are embedded the partial cDNA sequence and its flanking fragments.
• the flanking fragments are isolated by PCR with primers complementary to the known vector and cDNA sequences and then sequenced directly.
• the DNA sequences from these fragments plus the original partial cDNA sequence are assembled into a continuous fragment, resulting in the extension of the partial cDNA sequence and the eventual determination of its full-length gene sequence by repeating the process, if necessary, until a complete open reading frame is obtained.
• the fundamental principle of this process is to subdivide a complex library into superpools of about 10,000 to about 20,000 clones.
• a library of two million primary clones, a number large enough to cover most mRNA transcripts expressed in the tissue, can be subdivided into 188 pools and stored in two 96-well plates. Since the number of transcripts for most genes is fewer than one copy per ⁇ 10,000 transcripts in total cellular mRNA, each pool is unlikely to contain more than one clone for a given cDNA sequence. Such reduced complexity makes it possible to use PCR to isolate flanking fragments of partial cDNA sequences larger than those obtained by known methods.
• multiple primer combinations from an EST or other partial cDNA sequence, in combination with flanking vector primer oligonucleotides can be used to "walk" in both directions away from the internal, gene specific, sequence, and respective primers, such that a contig representing a full length cDNA can be constructed.
• This procedure relies on the ability to screen multiple pools which comprise a representative portion of the total cDNA library. This procedure is not dependent upon using a cDNA library with directionally cloned inserts.
• both ⁇ ' and 3' vector and gene specific primers are added and a contig map is constructed from additional screening of positive pools using both vector primers and gene specific primers.
• these gene spedfic primers are initially constructed from a known nucleic acid fragment such as an expressed sequence tag.
• gene spedfic primers are utilized from the ⁇ ' and 3' boundaries of the newly identified regions of the cDNA.
• the vector orientation of a yet unidentified fragment be known. Instead, all combinations are tested on a positive pool and the actual vector orientation is determined by the ability of certain vector/gene specific primers to generate the predicted PCR fragment.
• a full-length cDNA can then be easily constructed by known subcloning procedures.
• the AOMFO ⁇ gene from different spedes are isolated by screening of a cDNA library with portions of the gene that have been obtained from cDNA of the spedes of interest using PCR primers designed from the human AOMFO ⁇ sequence.
• Degenerate PCR is performed by designing primers of 17-20 nucleotides with 32-128 fold degeneracy by selecting regions that code for amino acids that have low codon degeneracy e.g. Met and Trp. When selecting these primers preference is given to regions that are conserved in the protein.
• PCR products are analyzed by DNA sequence analysis to confirm their similarity to human AOMFO ⁇ . The correct product is used to screen cDNA libraries by colony or plaque hybridization at high stringency.
• probes derived directly from the human AOMFO ⁇ gene are utilized to isolate the cDNA sequence of AOMFO ⁇ from different spedes by hybridization at reduced stringency.
• a cDNA library can be generated as known in the art or as described herein.
• transgenes are genetic construct including a gene.
• the transgene is integrated into one or more chromosomes in the cells in an animal or its ancestor by methods known in the art. Once integrated, the transgene is carried in at least one place in the chromosomes of a transgenic animal.
• a gene is a nucleotide sequence that encodes a protein.
• the gene and/or transgene can also include genetic regulatory elements and/or structural elements known in the art.
• animal is used herein to include all mammals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages. Preferably the animal is a rodent, and most preferably mouse or rat.
• a "transgenic animal” is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus. This introduced DNA molecule can be integrated within a chromosome, or it can be extra-chromosomally replicating DNA.
• transgenic animal refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If offspring in fact possess some or all of the genetic information, then they, too, are transgenic animals.
• the genetic information is typically provided in the form of a transgene carried by the transgenic animal.
• the genetic information received by the non-human animal can be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual redpient. In the last case, the information can be altered or it can be expressed differently than the native gene. Alternatively, the altered or introduced gene can cause the native gene to become non-functional to produce a "knockout" animal.
• a "targeted gene” or “Knockout” (KO) transgene is a DNA sequence introduced into the germline of a non- human animal by way of human intervention, including but not limited to, the methods described herein.
• the targeted genes of the invention include nucleic acid sequences which are designed to specifically alter cognate endogenous alleles of the non-human animal.
• An altered AOMFO ⁇ receptor gene should not fully encode the same receptor endogenous to the host animal, and its expression product can be altered to a minor or great degree, or absent altogether.
• the altered AOMFO ⁇ gene induce a null, "knockout,” phenotype in the animal.
• a more modestly modified AOMFO ⁇ gene can also be useful and is within the scope of the present invention.
• ES cells can be obtained from pre- implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al, Nature 292:l ⁇ 4-l ⁇ 6 (1981); Bradley et al, Nature 309:2 ⁇ -2 ⁇ 8 (1984); Gossler et al Proc. Natl. Acad. Sd. USA 83:906 ⁇ -9069 (1986); and Robertson et al, Nature 322:44 ⁇ -448 (1986)).
• Transgenes can be efficiently introduced into the ES cells by a variety of standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction.
• the resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
• the introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (R. Jaenisch, Sdence 240: 1468-1474 (1988)). Animals are screened for those resulting in germline transformants. These are crossed to produce animals homozygous for the transgene.
• Methods for evaluating the targeted recombination events as well as the resulting knockout mice are readily available and known in the art. Such methods include, but are not limited to DNA (Southern) hybridization to detect the targeted allele, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots to detect DNA, RNA and protein.
• DNA Southern
• PCR polymerase chain reaction
• PAGE polyacrylamide gel electrophoresis
• Western blots to detect DNA, RNA and protein.
• the presence of a mutant, allele or variant sequence within cells of an organism, particularly when in place of a homologous endogenous sequence, may allow the organism to be used as a model in testing and/or studying the role of the AOMFO ⁇ gene or substances which modulate activity of the encoded polypeptide and/or promoter in vitro or are otherwise indicated to be of therapeutic potential.
• the present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic add molecules disclosed throughout this specification. Therefore, the present invention also relates to methods of expressing AOMFO ⁇ and biological equivalents disclosed herein, assays employing these recombinantly expressed gene products, cells expressing these gene products, and modulators, agonistic and/or antagonistic compounds identified through the use of assays utilizing these recombinant forms, including, but not limited to, one or more compounds or molecules that act through direct contact with the receptor, particularly with the ligand binding domain, or through direct or indirect contact with a ligand which either interacts with the receptor or with the transcription or translation of AOMFO ⁇ , thereby modulating AOMF05 expression.
• a variety of expression vectors can be used to express recombinant AOMFO ⁇ in host cells.
• Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host.
• Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells and animal cells. Spedfically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria- animal cells.
• An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
• a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
• a strong promoter is one which causes mRNAs to be initiated at high frequency.
• Expression vectors can include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
• mammalian expression vectors which can be suitable for recombinant human AOMFO ⁇ expression, include but are not limited to, pcDNA3.1 (Invitrogen), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Biolabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG ⁇ (Stratagene), EBO-pSV2-neo (ATCC 37 ⁇ 93) pBPV- 1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2- dhfr (ATCC 37146), pUCTag (ATCC 37460), and lZD3 ⁇ (AT
• a variety of bacterial expression vectors can be used to express recombinant human AOMFO ⁇ in bacterial cells.
• Commercially available bacterial expression vectors which are suitable for recombinant human AOMFO ⁇ expression include, but are not limited to pQE (Qiagen), pETlla (Novagen), lambda gtll (Invitrogen), and pKK223- 3 (Pharmacia).
• a variety of fungal cell expression vectors can be used to express recombinant human AOMFO ⁇ in fungal cells.
• Commercially available fungal cell expression vectors which are suitable for recombinant human AOMFO ⁇ expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
• insect cell expression vectors can be used to express recombinant receptor in insect cells.
• Commercially available insect cell expression vectors which are suitable for recombinant expression of human AOMFO ⁇ include but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).
• An expression vector containing DNA encoding a human AOMFO ⁇ -like protein can be used for expression of human AOMFO ⁇ in a recombinant host cell.
• Recombinant host cells can be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, pordne, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines.
• L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-8 ⁇ ), 293 (ATCC CRL l ⁇ 73), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 16 ⁇ 0), COS-7 (ATCC CRL 16 ⁇ l), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL
• HeLa ATCC CCL 2
• C127I ATCC CRL 1616
• BS-C-1 ATCC CCL 26
• MRC- ⁇ ATCC CCL 171
• CPAE ATCC CCL 209
• the expression vector can be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation.
• the expression vector-containing cells are individually analyzed to determine whether they produce human AOMFO ⁇ protein. Identification of human AOMFO ⁇ expressing cells can be done by several means, including but not limited to immunological reactivity with anti- human AOMFO ⁇ antibodies, labeled ligand binding and the presence of host cell-assodated human AOMFO ⁇ activity.
• the cloned human AOMFO ⁇ cDNA obtained through the methods described herein can be recombinantly expressed by molecular cloning into an expression vector (such as pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28) containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant human AOMFO ⁇ .
• an expression vector such as pcDNA3.1, pQE, pBlueBacHis2 and pLITMUS28
• Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as effidently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
• cDNA molecules including but not limited to the following can be constructed: a cDNA fragment containing the full-length open reading frame for human AOMFO ⁇ as well as various constructs containing portions of the cDNA encoding only specific domains of the protein or rearranged domains of the protein. All constructs can be designed to contain none, all or portions of the ⁇ ' and/or 3' untranslated region of a human AOMFO ⁇ cDNA.
• the expression levels and activity of human AOMFO ⁇ can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells.
• this AOMFO ⁇ cDNA construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells.
• AOMFO ⁇ polypeptides can be recovered.
• AOMFO ⁇ protein purification procedures are available and suitable for use.
• AOMFO ⁇ protein and polypeptides can be purified from cell lysates and extracts, or from conditioned culture medium, by various combinations of, or individual application of methods including ultrafiltration, acid extraction, alcohol predpitation, salt fractionation, ionic exchange chromatography, phosphocellulose chromatography, lecithin chromatography, affinity (e.g., antibody or His-Ni) chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and chromatography based on hydrophobic or hydrophillic interactions.
• protein denaturation and refolding steps can be employed.
• High performance liquid chromatography (HPLC) and reversed phase HPLC can also be useful. Dialysis can be used to adjust the final buffer composition.
• Anti-AOMFO ⁇ Antibodies The present invention also relates to polyclonal and monoclonal antibodies raised in response to either the human form of AOMF05 disclosed herein, or a biologically active fragment thereof. It will be espedally preferable to raise antibodies against epitopes within the NH2-terminal domain or the extracellular inter-membrane domains of AOMFO ⁇ . It is also preferable to raise antibodies to epitopes which show the least homology to other known glycoprotein hormone receptor proteins.
• An antibody is specific for an AOMFO ⁇ epitope if one of skill in the art can use standard techniques to determine conditions under which one can detect AOMFO ⁇ in a Western Blot of a sample from a host cell that displays AOMFO ⁇ on its surface.
• the blot can be of a native or denaturing gel as appropriate for the epitope.
• An antibody is highly spedfic for an AOMFO ⁇ epitope if no nonspedfic background binding is visually detectable.
• An antibody can also be considered highly specific for AOMFO ⁇ if the binding of the antibody to AOMF05 can not be competed by non-AOMF05 peptides, polypepetides or proteins.
• Recombinant AOMFO ⁇ protein can be separated from other cellular proteins by use of an immuno affinity column made with monoclonal or polyclonal antibodies spedfic for full-length AOMFO ⁇ protein, or polypeptide fragments of AOMFO ⁇ protein. Additionally, polyclonal or monoclonal antibodies can be raised against a synthetic peptide (usually from about 9 to about 2 ⁇ amino adds in length) from a portion of the protein as disclosed in SEQ ID NO:2. Monospecific antibodies to human AOMFO ⁇ are purified from mammalian antisera containing antibodies reactive against human AOMFO ⁇ or are prepared as monoclonal antibodies reactive with human AOMFO ⁇ using the technique of Kohler and Milstein (1975, Nature 256: 495-497).
• Monospecific antibody as used herein is defined as a single antibody spedes or multiple antibody species with homogenous binding characteristics for human AOMFO ⁇ .
• Homogenous binding refers to the ability of the antibody spedes to bind to a spedfic antigen or epitope, such as those assodated with human AOMFO ⁇ , as described herein.
• Human AOMFO ⁇ -spedfic antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with an appropriate concentration of human AOMFO ⁇ protein or a synthetic peptide generated from a portion of human AOMFO ⁇ with or without an immune adjuvant.
• Preimmune serum is collected prior to the first immunization.
• Each animal receives between about 0.1 mg and about 1000 mg of human AOMFO ⁇ protein associated with an acceptable immune adjuvant.
• acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-predpitate, water in oil emulsion containing Corynebacterium parvum and tRNA.
• the initial immunization consists of human AOMF05 protein or peptide fragment thereof in, preferably, Freund's complete adjuvant at multiple sites either subcutaneously (SC), intraperitoneally (IP) or both.
• SC subcutaneously
• IP intraperitoneally
• Each animal is bled at regular intervals, preferably weekly, to determine antibody titer.
• the animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of human AOMFO ⁇ in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about -20°C. Monoclonal antibodies (mAb) reactive with human
• AOMFO ⁇ are prepared by immunizing inbred mice, preferably Balb/c, with human AOMFO ⁇ protein.
• the mice are immunized by the IP or SC route with about 1 mg to about 100 mg, preferably about 10 mg, of human AOMF05 protein in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed herein. Freund's complete adjuvant is preferred.
• the mice receive an initial immunization on day 0 and are rested for about 3 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 1 to about 100 mg of human AOMF05 in a buffer solution such as phosphate buffered saline by the intravenous (IV) route.
• IV intravenous
• Lymphocytes from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art.
• Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. Fusion partners can include, but are not limited to: mouse myelomas P3/NSl/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred.
• the antibody produdng cells and myeloma cells are fused in polyethylene glycol, about 1000 mol. wt., at concentrations from about 30% to about ⁇ 0%.
• Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected form growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay such as solid phase immunoradioassay (SPIRA) using human AOMFO ⁇ as the antigen. The culture fluids are also tested in the Ouchterlony predpitation assay to determine the isotype of the mAb.
• SPIRA solid phase immunoradioassay
• Hybridoma cells from antibody positive wells are cloned by a technique such as the soft agar technique of MacPherson, 1973, Soft Agar Techniques, in Tissue Culture Methods and Applications, Kruse and Paterson, Eds., Academic Press.
• Monoclonal antibodies are produced in vivo by injection of pristine primed Balb/c mice, approximately O. ⁇ ml per mouse, with about 2 x 10 6 to about 6 x 10 6 hybridoma cells about 4 days after priming. Asdtes fluid is collected at approximately 8-12 days after cell transfer and the monoclonal antibodies are purified by techniques known in the art.
• In vitro production of anti-human AOMFO ⁇ mAb is carried out by growing the hybridoma in DMEM containing about 2% fetal calf serum to obtain suffident quantities of the specific mAb.
• the mAb are purified by techniques known in the art.
• Antibody titers of asdtes or hybridoma culture fluids are determined by various serological or immunological assays which include, but are not limited to, predpitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of human AOMFO ⁇ in body fluids or tissue and cell extracts.
• serological or immunological assays include, but are not limited to, predpitation, passive agglutination, enzyme-linked immunosorbent antibody (ELISA) technique and radioimmunoassay (RIA) techniques. Similar assays are used to detect the presence of human AOMFO ⁇ in body fluids or tissue and cell extracts.
• Human AOMFO ⁇ antibody affinity columns are made, for example, by adding the antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. The remaining activated esters are then quenched with 1M ethanolamine HC1 (pH 8). The column is washed with water followed by
• AOMFO ⁇ -spedfic affinity beads or AOMFO ⁇ -specific antibodies are used to isolate 35 S-methionine labeled or unlabelled AOMFO ⁇ .
• Labeled AOMFO ⁇ protein is analyzed by SDS- PAGE.
• Unlabelled AOMFO ⁇ protein is detected by Western blotting, ELISA or RIA assays employing either AOMFO ⁇ protein spedfic antibodies and/or antiphosphotyrosine antibodies.
• the present invention is also directed to methods for screening for compounds or molecules which modulate the expression of DNA or RNA encoding a human AOMFO ⁇ protein.
• Compounds or molecules which modulate these activities can be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules. They can modulate by increasing or attenuating the expression of DNA or RNA encoding human AOMFO ⁇ .
• Compounds that modulate the expression of DNA or RNA encoding human AOMFO ⁇ or are agonists or antagonists of the biological function thereof can be detected by a variety of assays.
• the assay can be a simple "yes/no" assay to determine whether there is a change in expression or function.
• kits containing human AOMFO ⁇ , antibodies to human AOMFO ⁇ , or modified human AOMFO ⁇ can be prepared by known methods for such uses.
• the DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention can be used to screen and measure levels of human AOMFO ⁇ .
• the recombinant proteins, DNA molecules, RNA molecules and antibodies lend themselves to the formulation of kits suitable for the detection and typing of human AOMFO ⁇ .
• Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container.
• the carrier would further comprise reagents such as recombinant AOMFO ⁇ or anti- AOMFO ⁇ antibodies suitable for detecting human AOMFO ⁇ .
• the carrier can also contain a means for detection such as labeled antigen or enzyme substrates or the like.
• compositions comprising agonists, antagonist or modulators of human AOMFO ⁇ can be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation can be found in Remington's Pharmaceutical Sdences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, DNA, RNA, modified human AOMFO ⁇ , or either AOMFO ⁇ modulators, agonsits or antagonists.
• compositions of the invention are administered to an individual in amounts suffident to treat or diagnose disorders.
• the effective amount can vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
• compositions can be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
• chemical derivative describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties can improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties can attenuate undesirable side effects of the base molecule or decrease the toxidty of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sdences.
• the present invention also provides a means to obtain suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the methods of treatment of the present invention.
• the compositions containing compounds or molecules identified according to this invention as the active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
• the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
• compounds of the present invention can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily.
• compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
• the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
• the active agents can be administered concurrently, or they each can be administered at separately staggered times.
• the dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, spedes, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular compound thereof employed.
• a physidan or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
• Polypeptide sequences of human G-protein coupled glycoprotein hormone receptors were used as probes to search the EST database dbEST of NCBI (National Center for Biotechnology Information) using the search program tFASTA.
• the sequences chosen were the protein sequences of known human receptors, i.e., receptors for FSH (Follicle- stimulating hormone), TSH (thyroid-stimulating hormone), LH (leutinizing hormone).
• An EST (GenBank accession #T73957) was found to encode a polypeptide that is approximately 30% identical to these receptors at the amino acid level.
• primers were used for the isolation of AOMF05 as described below. For convenience and clarity, the SEQ ID NOS are presented here. In the following description, primers can be referred to by the numerical component of their designation.
• AOMF05 The full-length sequence of AOMF05 was isolated from a fetal brain cDNA library by multiple rounds RCCA (Reduced Complexity cDNA Analysis, described herein).
• RCCA Reduced Complexity cDNA Analysis, described herein.
• primers predicted to be specific for the AOMF05 EST T73957 (primers F30 and Rl 17), as well as oligonucleotide primers both 5' and 3' of the poly linker sequence of the vector (primers PBS.873F and PBS.543R) were used.
• PCR reactions were carried out with Amplitaq Gold (Perkin Elmer-Roche, Branchberg, NJ, U.S. A) using standard PCR conditions as suggested by the enzyme supplier.
• nested insert- vector PCRs were carried out on the positive pools with the following combinations: primary reactions, F30+PBS.543R, F30+ PBS.873F; Rl 17+ PBS.543R, Rl 17+ PBS.873F. Secondary (nested) reactions, F77+ PBS.578R, F77+ PBS.838F, R71+ PBS.578R, R71+ PBS.838F. PCR products were then sequenced and assembled. Two new sequencing primers R126s and F1803s for the 3' and 5' direction were synthesized and used to sequence the previous nested PCR products. The assembled sequence contained an open reading frame.
• the sequence containing the open reading frame was amplified using two primers F16 and R2289 and cloned into the vector pCR2.1
• a walking primer R175 for the 5' direction was synthesized.
• This primer and vector specific primer PBS.538R was used to scan the superpooled libraries. After positive rows were identified 5' extension was performed on these rows and the product sequenced and assembled. From the new sequence two primers F31 and R167 were picked to identify new pools in the fetal brain and prostrate cDNA libraries. After positive pools were identified, 5' extension was carried with the following primer combinations: R167+ PBS 578R, R167+ PBS.838F. PCR products were then sequenced and assembled into the contig. Based on the new sequence, another 5' primer R69 was synthesized.
• This primer was then used to amplify with PBS.838F or PBS.543R on the positive pools in the presence of 5% DMSO.
• the PCR products were then sequenced and assembled into a single contig. This sequence contains an open reading frame of 2850 base pairs, encoding a polypeptide of 949 amino acids.
• CNheMF05-2479 were synthesized and used to amplify the 5' end.
• the PCR fragment was digested with Nhel and ligated with Nhel-digested pMF053.1.A.
• the resulting plasmid was verified by physical mapping and sequencing, and named pcDNA3.1MF05.
• FIGS. 1 A-1B SEQ ID NO:l
• FIGS. 4A-4C SEQ ID NO:3.
• the amino acid sequence of the variants of this receptor are provided in FIG. 2 (SEQ ID NO:2) and FIG. 5 (SEQ ID NO:4).
• FASTA searches and phylogenetic analysis were performed using the program Pepplot of GCG (Genetics Computer Group, Madison, Wisconsin, USA). The analysis revealed that AOMF05 is a member of the G-protein coupled glycoprotein hormone receptor family. Hydropathy analysis was performed using the program
• AOMF05 has 7 transmembrane domains typical of the rhodopsin family of G-protein coupled receptors. The domains begin at about amino acid 539 of SEQ ID NO:2 or 4.
• the deduced polypeptide sequence of AOMF05 contains several sites for cleavage of a signal peptide from the N-terminus of the protein (FIG. 7).
• Multi-tissue Northern blot analysis was performed as follows. Ready-to-use human multi-tissue Northern blots were purchased from Clontech (Clontech, Palo Alto, CA, USA). A total of six blots were used to analyze the expression of AOMF05 in human tissues.
• Fragments of the AOMF05 cDNA were labeled with 32 P by random priming using the REDDY-PRIME® labeling kit (Amersham, Inc., Chicago, IL, USA). Reactions were carried using the protocol of the kit supplier. Approximately 50 ng of DNA in 45 ⁇ l of H 2 0 was boiled for 3 minutes., and then quickly chilled to 0°C for 5 minutes. The DNA solution was transferred to REDDY-PRIME® tube and mixed with the lyophilized reagents in the tube. Then, 5.0 ⁇ l of ⁇ - 32 P-dCTP (-5000 Ci/mM) was added and the tube was incubated at 37°C for 15 minutes. The reaction was stopped by adding 5.0 ⁇ l of 0.5 M EDTA (pH8.0). Unincorporated nucleotides were removed by gel-filtration using a spun column.
• the labeled fragments were used as probes for AOMF05 RNA.
• Hybridizations were carried out in the ExpressHyb buffer of Clontech following the protocol provided by the membrane supplier Clontech (Palo Alto, CA, USA). The membranes were prehybridized at 68°C for 1 hr in the Expresshyb buffer with gentle agitation. The 32 P-labeled probe was denatured by adding NaOH to a final concentration of 0.2 nM and then added into the hybridization solution. Hybridizations were performed for 3 hours at 68°C. The membranes were removed from the hybridization buffer and washed once in 2x SSC, 0.1% SDS, for 10 min. at room temperature. The membranes were then washed at 0.1 xSSC, 0.1 % SDS for 30 minutes at 50°C. The blots were analyzed using a Phosphaimager (Molecular Dynamics, Sunnyvale, CA, USA).
• AOMF05 was most abundantly expressed in pancreas and moderately expressed in heart, brain, liver, kidney, skeletal muscle, placenta, adrenal medulla, adrenal cortex, thyroid, stomach, and testis (FIG. 8). In all of these tissues, AOMF05 was detected as a transcript of ⁇ 5.5 kb, except in placenta where an additional ⁇ 4.5 kb messenger was also detected.
• the AOMFO ⁇ cDNA is used as a probe to isolate human genomic DNA encoding the receptor.
• the cDNA can be used in its entirety or portions of the sequence can be used. If portions of the sequence less than 100 nucleotides are used as a probe, one should perform homology analysis of the selected probe sequence against human sequences in general to assess the uniqueness of the chosen sequence in human DNA. If the chosen sequence exhibits high homology to a variety of human DNA sequences, then that sequence will not perform well as a probe spedfic for AOMFO ⁇ genomic DNA. For example, portions of the cDNA encoding amino acid sequences that are highly conserved among G-protein coupled receptors can be used.
• the probe is labeled by any means known in the art, including but not limited to incorporation of radioisotopes or biotin.
• the probe is hybridized against a library of human genomic DNA fragments.
• the stringency of the hybridization reaction can be adjusted by means known in the art, e.g., varying salt concentrations and temperature, to obtain appropriately spedfic hybridization of the probe to the target sequence.
• the fragments identified by the probe can be sequenced or subjected to restriction enzyme digestion to confirm that they contain AOMFO ⁇ genomic DNA.
• the entire genomic gene may not be contained within any one identified fragment. In that case, one will be required to perform chromosome walking, e.g. , using an identified fragment as a probe to isolate additional fragments that overlap in the chromosome, to isolate the entire gene. If the isolation of overlapping fragments is required, one can use known methods of manipulation of DNA to construct a contiguous DNA fragment encoding the entire AOMF05 genomic DNA.
• a polynucleotide having an AOMFO ⁇ nucleotide sequence e.g., the nucleotide sequence of a cDNA or genomic DNA encoding a full length AOMFO ⁇ receptor, or a polynucleotide encoding a partial sequence of the receptor, sequences flanking the coding sequence, or both, can be combined into a vector for the integration of the polynucleotide into the genome of an animal.
• the AOMFO ⁇ sequence can be from a human AOMFO ⁇ or from the animal's AOMFO ⁇ .
• the target cell for transgene introduction is a murine embryonic stem cell (ES).
• ES cells can be obtained from pre- implantation embryos of a variety of non-human animals cultured in vitro and fused with embryos (M. J. Evans et al, Nature 292:l ⁇ 4-l ⁇ 6
• the transgene is introduced into the murine ES cells by microinjection, however, a variety of standard techniques such as DNA transfection, or retrovirus-mediated transduction can be used.
• the injected ES cells are then combined with blastocysts from a non-human animal.
• the introduced ES cells colonize the embryo and contribute to the germ line of the resulting chimeric animal (R. Jaenisch, Sdence 240: 1468-1474 (1988)).
• the chimeric mice are screened for individuals in which germline transformation has occurred. These are crossed to produce animals homozygous for the transgene.
• the targeted recombination events as well as the resulting mice are evaluated by techniques well known in the art, including but not limited to DNA (Southern) hybridization to detect the targeted allele, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots to detect DNA, RNA and protein.
• DNA Southern
• PCR polymerase chain reaction
• PAGE polyacrylamide gel electrophoresis
• Western blots to detect DNA, RNA and protein.
• transgenic animals Three basis types of transgenic animals are created depending on the construction of the transgene vector. If the vector is designed to include a nucleotide sequence that encodes a full length human AOMFO ⁇ receptor and to integrate at a site other than the animal's endogenous AOMFO ⁇ gene, the resultant transgenic animal will express both a native and human AOMFO ⁇ receptors. If the vector is designed without a cognate AOMFO ⁇ gene and to integrate at the site of the animal's endogenous AOMFO ⁇ gene such that after integration the endogenous gene is altered to such an extent that the animal lacks a functional AOMFO ⁇ receptor, then a knockout animal is produced.
• the resultant animal lacks a native AOMFO ⁇ receptor and expresses a human AOMFO ⁇ receptor.
• Animals having a human gene and lacking an endogenous gene can also be created by crossing the first type of animal with a knockout animal to obtain animals homozygous for the knockout and homozygous for the added human AOMFO ⁇ gene. This can be fadlitated if the human gene integrates in a chromosome different from the chromosome carrying the endogenous AOMFO ⁇ gene.
• Transgenic animals are a source of cells and tissues for use in assays of AOMFO ⁇ modulation, activation or inhibition. Cells can be removed from the animals, established as cell lines and maintained in culture as convenient. EXAMPLE 6
• Glutathione S-transferase GST
• Polypeptide fusion constructs are made by inframe fusion of all or a portion of the N-terminal ligand-binding domain of the AOMFO ⁇ G-protein coupled glycoprotein hormone receptor and the carboxy terminus of the GST gene.
• the disclosure of SEQ ID NOS: 1-4 allow the artisan of ordinary skill to construct any such nucleic acid molecule encoding a GST- AOMFO ⁇ fusion protein.
• fusions can be constructed using a polynucleotide that encodes the N-terminal fragment of AOMFO ⁇ from about amino add 20 to about ⁇ 39, or from about 20 to the end of the sequence of SEQ ID NO:2, fused to GST C- terminus.
• Soluble recombinant AOMFO ⁇ fusion proteins can be expressed in various expression systems, some of which are described herein, including Spodoptera frugiperda (Sf21) insect cells using a baculovirus expression vector (e.g., Bac-N-Blue DNA from Invitrogen or pAcG2T from Pharmingen). The fusion protein is then loaded onto a glutathione column.
• the C-terminal domain of GST binds to the glutathione and the N-terminal region of AOMFO ⁇ is exposed to the buffer phase.
• a sample that may contain a ligand of the AOMFO ⁇ receptor is passed over the column.
• the sample can be cell or tissue extracts, bodily fluids or compounds or molecules that are purified or synthesized.
• the sample can be applied directly or after dilution or dialysis in a buffer approximating physiological conditions.
• Ligands of the receptor are bound by the N-terminal domain of AOMFO ⁇ .
• the ligands are eluted. This can be achieved, for example, by applying a gradient of NaCl to the column in wash buffer.
• Unknown ligands present in biological extracts or fluids can be characterized by standard chemical and biochemical methods.
• Ligands identified in this method can be used as candidates in assays for agonists or antagonists of the AOMFO ⁇ receptor.
• Assays for ligands can also be conducted as described below for assays for agonist and antagonists of AOMFO ⁇ .
• a candidate compound or molecule that shows agonist or antagonist activity can also be a ligand for AOMFO ⁇ .
• a polynucleotide of the present invention is used to transform or transfect the appropriate cells, or cells can be obtained and cultured from an appropriate transgenic animal.
• melanophores are transfected to express the AOMFO ⁇ G-protein coupled receptor.
• the transformed melanophores are exposed to both an activating ligand and a candidate compound. Inhibition of the signal generated by the Hgand indicates that the candidate is a potential antagonist of the receptor.
• the cells are contacted with candidate compounds and it is determined whether any compound activates the receptor to generate a signal. Activation of the receptor indicates that the candidate is a potential agonist of the receptor.
• Yeast expressing mammalian adenylate cyclase Yeast expressing mammalian adenylate cyclase.
• yeast that express mammalian adenylate cyclase are described in WO 9 ⁇ /30012, published November 9, 199 ⁇ . These yeast can be engineered to co-express the AOMFO ⁇ receptor in the presence of an appropriate G-protein.
• the transformed yeast are exposed to both an activating ligand of AOMFO ⁇ and a candidate compound. Inhibition of the signal generated by the ligand indicates that the candidate is a
• the cells are contacted with candidate compounds and it is determined whether any compound activates the receptor to generate a signal. Activation of the receptor indicates that the candidate is a potential agonist of the receptor.
• Yeast cells engineered to produce pheromone system protein surrogates can be used to screen for the ability of the surrogate to substitute for the cognate yeast pheromone receptor as described in WO 94/2302 ⁇ , published October 13, 1994.
• the method involves expressing the AOMFO ⁇ G-protein coupled receptor in Saccharomyces cerevisiae in which the receptor is linked to pheromone pathway.
• the yeast Ga subunit is generally deleted and replaced with a mammalian Ga protein so that the mammalian G protein-coupled receptor can be coupled to the yeast pheromone pathway.
• Members of a plasmid library capable of expressing peptides of random sequences are introduced into an appropriate yeast strain.
• Clones encoding agonist ligands for the AOMFO ⁇ receptor can be selected for their stimulation of the pheromone pathway.
• Clones encoding antagonist ligands for the AOMFO ⁇ receptor can be selected for their inhibition of the pheromone pathway in the presence of an AOMFO ⁇ agonist.
• libraries of chemicals can be screened for their agonist or antagonist activity by testing the chemicals directly.
• Another screening technique involves expressing the AOMFO ⁇ receptor wherein the receptor is linked to a phosphoHpase C or D.
• Cells including CHO, endothelial, embryonic kidney and other cells can be used.
• ligand and candidates are screened for agonist or antagonist activities by detecting the activation or inhibition or the receptor's activation of the phospholipase second signal.
• yeast cells expressing a heterologous phospholipase is found in WO 96/40939, published December 19, 1996.
• the yeast two-hybrid system expressing the AOMFO ⁇ G- protein coupled receptor can be used for screening for agonists and antagonists of the receptor (Fields and Song, 1989, Nature 340:24 ⁇ -246).
• the entire or portions of the extracellular domain of the G- protein coupled receptor can be fused to the DNA binding domain of transcription factor Gal4 or LexA.
• Yeast cells expressing these constructs are used to carry out screening for molecules that interact with the G-protein coupled receptor by using standard protocols such as those described previously (Fields and Song, 1989) of the two-hybrid screening method. Such molecules represent potential agonists or antagonists of the receptor.
• Compounds or molecules that are modulators of the receptor can be detected in assay described or as follows.
• An antibody specific for the extracellular domain of the receptor is obtained by standard techniques.
• the antibody can be polyclonal or monoclonal.
• the affinity of the antibody for the extracellular domain of the receptor should preferably be at least 10 6 , and more preferably at least 10 8 , to simplify conducting the assay.
• a cell culture that expresses the receptor is provided. The cell culture can be one that naturally expresses the
• Two samples of the culture are used in the assay.
• One sample is used as a control and is treated with a placebo, i.e., a compound or molecule determined to have no modulatory effects on the receptor in the assay.
• the second sample is treated with a candidate modulator.
• a portion of the culture can be withdrawn.
• the antibody can then be used to qualify or quantify the amount of receptor present on the surface of the cell. This can be done by numerous techniques known in the art including using antibody detectably labeled with 125 I, gold, enzyme or other known labels. .
• a detectable label can be carried on a second antibody specific for the first.
• the amount of receptor found on the cells treated with a potential modulator is quantitatively or qualitatively compared to the amount of receptor found on the control cells. A change in the former relative to the latter is indicative of the whether or not the test compound is a modulator of the receptor.
• the assay one can treat cells as described herein and then isolate the receptors present in treated and control cells.
• the receptor preparations can be made as crude cell extracts, membrane or intracellular fractions of the cells or after purification steps, e.g., chromatography, precipitation or affinity isolation steps. Crude, partially or highly purified preparations of receptors can be analyzed for receptor content, e.g. , by using antibodies spedfic for the receptor.
• any assay it can be advantageous to devise an internal control so that the results of different runs of assays can be compared to each other.
• a cellular protein that is unrelated to the receptor and present in relatively constant amounts in the cells used in the assay can serve as an internal control.
• the present invention includes methods of identifying compounds that specifically bind to an AOMF05 protein, as well as compounds identified by such methods.
• the specificity of binding of compounds having affinity for an AOMF05 protein is shown by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from these cells.
• Expression of the cloned receptor and screening for compounds that bind to an AOMF05 protein or that inhibit the binding of a known, radiolabeled ligand of AOMF05 to these cells, or membranes prepared from these cells provides an effective method for the rapid selection of compounds with high affinity for an AOMF05 protein.
• Such ligands need not necessarily be radiolabeled but can also be nonisotopic compounds that can be used to displace bound radiolabeled compounds or that can be used as activators in functional assays.
• Compounds identified by the herein method are likely to be agonists or antagonists of AOMF05 and may be peptides, proteins, or non-proteinaceous organic molecules.
• the present invention includes assays by which AOMF05 agonists and antagonists may be identified. Methods for identifying agonists and antagonists of other receptors are well known in the art and can be adapted to identify agonists and antagonists of AOMF05. Accordingly, the present invention includes a method for determining whether a candidate compound is a potential agonist or antagonist of AOMF05 that comprises: (a) transfecting cells with an expression vector encoding an
• the candidate compound is a potential agonist or antagonist of an AOMF05 protein.
• step (c) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
• the present invention also includes a method for determining whether a candidate compound is capable of binding to an AOMF05 protein, i.e., whether the candidate compound is a potential agonist or an antagonist of an AOMF05 protein, where the method comprises:
• test cells by transfecting cells with an expression vector that directs the expression of an AOMF05 protein in the cells; (b) exposing the test cells to the candidate compound;
• step (b) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
• the cells are eukaryotic cells.
• the cells are mammalian cells.
• the cells are L cells L-M(TK") (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86),
• the assays described herein can be carried out with cells that have been transiently or stably transfected with an AOMF05 protein.
• Transfection is meant to include any method known in the art for introducing an AOMF05 protein into the test cells.
• transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing an AOMF05 protein, and electroporation.
• binding of the candidate compound or agonist to AOMF05 can be measured by employing a labeled candidate compound or agonist.
• the candidate compound or agonist can be labeled in any convenient manner known to the art, e.g., radioactively, fluorescently, enzymatically.
• the AOMF05 protein has an amino acid sequence of SEQ ID NOS:2 or 4.
• the herein-described methods can be modified in that, rather than exposing the test cells to the candidate compound, membranes can be prepared from the test cells and those membranes can be exposed to the candidate compound.
• membranes can be prepared from the test cells and those membranes can be exposed to the candidate compound.
• Such a modification utilizing membranes rather than cells is well known in the art and is described in, e.g., Hess et al, 1992, Biochem. Biophys. Res. Comm. 184:260-268.
• the present invention provides a method for determining whether a candidate compound is capable of binding to an AOMF05 protein comprising:
• step (b) preparing membranes containing the AOMF05 protein from the test cells and exposing the membranes to a ligand of an AOMF05 protein under conditions such that the ligand binds to the AOMF05 protein in the membranes; (c) subsequently or concurrently to step (b), exposing the membranes from the test cells to a candidate compound;
• the present invention provides a method for determining whether a candidate compound is capable of binding to an AOMF05 protein comprising:
• Nucleic add according to the present invention e.g. encoding the authentic biologically active AOMFO ⁇ polypeptide or a functional fragment thereof, can be used in a method of gene therapy, to treat a patient who is unable to synthesize the active polypeptide or unable to synthesize it at the normal level, thereby providing the effect provided by the wild-type with the aim of treating and/or preventing one or more symptoms of one or more other diseases.
• Vectors such as viral vectors have been used to introduce genes into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a suffident proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide.
• the transfected nucleic add can be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
• viruses have been used as gene transfer vectors, including adenovirus, papovaviruses, such as SV40, vacdnia virus, herpesviruses, including HSV and EBV, and retroviruses, including gibbon ape leukemia virus, Rous Sarcoma Virus, Mandarin equine enchephalitis virus, Moloney murine leukemia virus and murine mammary tumorvirus.
• adenovirus papovaviruses
• papovaviruses such as SV40, vacdnia virus
• herpesviruses including HSV and EBV
• retroviruses including gibbon ape leukemia virus, Rous Sarcoma Virus, Kunststoffualian equine enchephalitis virus, Moloney murine leukemia virus and murine mammary tumorvirus.
• gibbon ape leukemia virus Rous Sarcoma Virus
• vacualian equine enchephalitis virus Moloney murine le
• Disabled virus vectors are produced in helper cell lines in which genes required for production of infectious viral particles are expressed.
• Helper cell lines are generally missing a sequence which is recognised by the mechanism which packages the viral genome and produce virions which contain no nucleic acid.
• a viral vector which contains an intact packaging signal along with the gene or other sequence to be delivered e.g. encoding the AOMFO ⁇ polypeptide or a fragment thereof
• Liposomes can encapsulate RNA, DNA and virions for delivery to cells. Depending on factors such as pH, ionic strength and divalent cations being present, the composition of liposomes can be tailored for targeting of particular cells or tissues. Liposomes include phospholipids and may include lipids and steroids and the composition of each such component can be altered. Targeting of liposomes can also be achieved using a spedfic binding pair member such as an antibody or binding fragment thereof, a protein, a sugar or a glycolipid.
• the aim of gene therapy using nucleic acid encoding the polypeptide, or an active portion thereof is to increase the amount of the expression product of the nucleic add in cells in which the level of the wild- type polypeptide is absent or present only at reduced levels.
• Such treatment can be therapeutic or prophylactic, particularly in the treatment of individuals known through screening or testing to have an AOMFO ⁇ allele associated with a disease state and hence a predisposition to the disease.
• Similar techiques can be used for anti-sense regulation of gene expression, e.g. targeting an antisense nucleic acid molecule to cells in which a mutant form of the gene is expressed, the aim being to reduce production of the mutant gene product.
• Other approaches to specific down-regulation of genes are well known, including the use of ribozymes designed to cleave specific nucleic acid sequences. Ribozymes are nucleic acid molecules, actually RNA, which specifically cleave single-stranded RNA, such as mRNA, at defined sequences, and their spedfidty can be engineered.
• Hammerhead ribozymes can be preferred because they recognize base sequences of about 11-18 bases in length, and so have greater specificity than ribozymes of the Tetrahymena type which recognise sequences of about 4 bases in length, though the latter type of ribozymes can also be useful in certain circumstances as will be recognized by one of skill in the art.
• References on the use of ribozymes include Marschall, et al. 1994. Cellular and Molecular Neurobiology 14( ⁇ ): ⁇ 23; Hasselhoff, 1988. Nature 334: ⁇ 8 ⁇ and Cech, 1988. J. Amer. Med. Assn. 260:3030.
• SEQ ID NOS:2 & 4 Two examples of the full length amino add sequence of the AOMFO ⁇ receptor protein is provided in SEQ ID NOS:2 & 4.
• nucleotide sequences can be DNA, RNA, mixtures of DNA and RNA or can include alternative linkages between nucleotides as described herein.

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